The subject matter disclosed herein relates to methods for isomerizing double bonds using chemical catalysts. Plants synthesize carotenoids, which are essential metabolites for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway for all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not known whether Z-ISO catalyzes isomerization alone or in combination with other enzymes.
Carotenoids constitute a large class of isoprenoids synthesized by all photosynthetic organisms, some bacteria, fungi and arthropods. Global vitamin A deficiency in children has sparked worldwide efforts to increase the levels of provitamin A carotenoids in food-crop staples. This goal rests on furthering knowledge of how plants control and biosynthesize carotenoids that can be converted in humans to vitamin A. Metabolic engineering and breeding of plants rich in particular carotenoids will continue to be an important objective for addressing the challenges of providing food security in a changing climate. Carotenoid functions are central to plant growth and development. The plant carotenoid-biosynthetic pathway is mediated by enzymes encoded in the nucleus and localized to chloroplasts or other plastids. The carotenoid-biosynthetic reactions begin with formation of the colorless 15-cis-phytoene, which undergoes desaturation and isomerization of double bonds to create carotenoids with yellow, red and orange colors. The pathway requires an electron-transfer chain and plastoquinones to channel electrons and protons produced during desaturation mediated by phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS). PDS produces 9,15,9′-tri-cis-ζ-carotene, which must be isomerized at the 15-15′-cis carbon-carbon double bond to form 9,9′-di-cis-ζ-carotene, the substrate for a second desaturase, ZDS (FIG. 1A). Although light can partially mediate this cis-trans carbon-carbon isomerization, Z-ISO is essential, especially in tissues receiving no light exposure, such as the endosperm tissue, which has been targeted for improvement of provitamin A carotenoids in efforts to alleviate global vitamin A deficiency. Plants with insufficient Z-ISO also grow poorly under the stress of fluctuating temperature. Because climatic variations alter the need for photosynthetic and nonphotosynthetic carotenoids, Z-ISO facilitates plant adaptation to environmental stress, a major factor affecting crop yield. Thus, Z-ISO is essential for maximizing plant fitness in response to environmental changes and for promoting accumulation of provitamin A carotenoids in edible tissues.
Mutants blocked in Z-ISO function accumulate 9,15,9′-tri-cis-ζ-carotene, the putative Z-ISO substrate. When the gene encoding Z-ISO is introduced into Escherichia coli cells producing 9,15,9′-tri-cis-ζ-carotene, this carotenoid is isomerized into the putative Z-ISO product, 9,9′-di-cis-ζ-carotene. These data suggest that Z-ISO is required for isomerization of the 15-cis bond in 9,15,9′-tri-cis-ζ-carotene but not the 15-cis bond in 15-cis-phytoene. In E. coli experiments, the isomerization activity associated with Z-ISO occurred in the presence of several upstream carotenoid-biosynthetic enzymes needed to produce the Z-ISO substrate. It is therefore desirable to determine whether Z-ISO is a bona fide enzyme that catalyzes isomerization through a unique mechanism requiring a redox-regulated heme cofactor.
The discussion above is merely provided for general background information and is not intended to be used as an aid in determining the scope of the claimed subject matter.